1. Structure and DNA Binding of the Human Rtf1 Plus3 Domain
Rob N. de Jong, Vincent Truffault, Tammo Diercks, Eiso AB, Mark A. Daniels, Rob Kaptein, Gert E. Folkers 
Structure 
Volume 16, Issue 1, Pages (January 2008)
DOI: /j.str
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2. Figure 1 The Structural Organization of the Rtf1 Plus3 Domain
Color coding: red, α helices; cyan, β strands; green, 310 helix. (A) Sequence alignment of ten proteins representative of the Plus3 domain sequence space. Rtf1_human (Swiss-Prot Q92541; top), Rtf1_yeast (S. cerevisiae P53064; bottom), and Swiss-Prot accession numbers for the other entries are indicated. Asterisks indicate the three conserved positively charged amino acids after which the Plus3 domain was named. Species from top to bottom: Petroselinum c., Neurospora c., Arabidopsis t., Schizosaccharomyces p., Oryza s., Drosophila m., Caenorhabditis e., and Drosophila m.. (B) Topology diagram of the novel Plus3 domain fold. (C) Superimposed NMR ensemble of the 25 lowest energy structures. Structural figures were created with PyMol (DeLano, 2002).
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3. Figure 2 The Solution Structure of the Rtf1 Plus3 Domain
(A) Two orthogonal ribbon presentations, rotated by 90° around the y axis.
(B) Stereoview of the backbone trace with every tenth Cα atom marked by a black sphere. Numbers indicate amino acid positions.
(C) The fully conserved residues R367, R389, and S444 cluster on the α/β subdomain interface (same orientation as [B]).
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4. Figure 3 Conserved Residues in the Rtf1 Plus3 Domain
For clarity, most hydrogens were omitted from the figures. (A) Core of the β subdomain. The Y416 OH group in the β hairpin hydrogen bonds to the L440 backbone amide proton and K425 backbone carbonyl group. (B) The hydrophobic core of the four-helical bundle, color coded as in Figure 1. Side chains colored according to the CPK scheme with carbon, gray; hydrogen, white; oxygen, red; nitrogen, blue. (C) The S. cerevisiae mutations V274D (Hs I390, in green) and (D) M289K (Hs V403, in green), which cause temperature-sensitive phenotypes, disrupt packing of the β sheet core and β sheet/α5 interface, respectively. Other amino acids are color coded by conservation scores, calculated by using ConSurf (Landau et al., 2005) with the alignment in Figure 1A as input, ranging from white (not conserved) to red (highly conserved).
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5. Figure 4
The Plus3 β Subdomain Displays Structural Similarity to Nucleic Acid Binding Proteins
The Rtf1 Plus3 domain is color coded as in Figure 2A, while α1, α2, and α6 have been omitted for clarity. Structural homologs retrieved and aligned with DALI (Holm and Sander, 1993) are displayed in dark blue, with discussed features highlighted in gold. (A) Overlay with the Argonaute PAZ domain (PDB code: 1T2R) complexed to a pentanucleotide RNA, of which two are visible in the structure (gold) (Lingel et al., 2004). (B) Overlay with the bacterial transcription elongation factor NusG (1M1G), with the KOW motif highlighted in gold (Steiner et al., 2002). (C) Sequence similarity between the KOW motif in transcription elongation factors and the Rtf1 Plus3 domain. Spt5 from Saccharomyces cerevisiae (Spt5_Sc) and Homo sapiens (Spt5_Hs) contain multiple KOW motifs numbered from N to C terminus. RFAH_Ec: Escherichia coli transcriptional activator rfaH. NusG_Aa: Aquifex aeolicus transcription antitermination protein NusG. RL24_Aa: Aquifex aeolicus 50S ribosomal protein L24. Yeast and human Rtf1 are indicated at the bottom. Asterisks indicate the position of thermosensitivity causing mutations with elongation phenotypes (Figures 2C and 2D).
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6. Figure 5 The Rtf1 Plus3 Domain Binds Single-Stranded DNA
(A) Electromobility shift assays (EMSA) with a double-stranded, single-stranded, and bubble DNA consisting of two ds stems separated by two opposing unpaired ten nucleotide single strands. From left to right, stepwise two-fold increasing concentrations up to 6 μM of Rtf1 Plus3 were incubated with DNA; “-” indicates no protein. Closed arrowheads indicate DNA/protein complex.
(B) EMSA with the bubble DNA substrate as in Figure 5A but with increasing amounts of His-GST-Rtf1 Plus3 (open arrowhead) and a His-Rtf1 Plus3 control (closed arrowhead).
(C) EMSA with bubble DNA and 3 or 6 μM His-Rtf1 Plus3 incubated with an α-His HRP conjugate that binds to the His-tag. The closed arrowhead indicates DNA/His-Rtf1 Plus3 complex, the open arrowhead the ternary DNA/protein/α-His HRP conjugate complex.
(D) EMSA with bubble DNA and 3μM His-Rtf1 Plus3 complex in the absence (+) or presence of 0.008, 0.04, 0.2, 1 μM of the indicated oligonucleotides as competitor: ss, BCTC; fork, splayed arm with 10 bp dsDNA and 10 unpaired nucleotides (dT10); hairpin, stem-loop substrate with 20 unpaired bases (dT20); ds, 20 bp dsDNA.
(E) Compound chemical shift deviations in ppm for 15N-bound protons (deviation = [(6.5 × δHN)2 + (δN)2]1/2) upon addition of 100 μM bubble DNA. The dotted line indicates the minimal threshold chosen for the color scale in Figure 6. Secondary structure elements are indicated below the figure, colored as in Figure 1C. Missing bars indicate unobservable protons for which the remaining signals were too weak at this protein concentration (100 μM) or disappeared upon DNA addition.
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7. Figure 6 Mapping of Rtf1 Plus3 Amino Acids Implicated in ssDNA Binding
(A) Ribbon presentation of Plus3 domain, oriented as in Figure 3. Chemical shift perturbation is color coded from white (below threshold of 0.12 ppm) to red (maximal). Grey indicates missing or disappearing signals during titration.
(B) SDS-PAGE of purified Plus3 mutants. Approximately 1 μg of single-column purified His-tagged mutant proteins were separated by SDS-PAGE and stained by Coomassie brilliant blue. WT, wild-type Rtf1 Plus3.
(C) EMSA of 0.8 μM Rtf1 Plus3 proteins on the bubble ss/ds DNA probe as described in Figure 5. (-) indicates extract purified from empty vector. A filled arrowhead indicates the position of the Plus3/DNA complex.
Structure  , DOI: ( /j.str )
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